CN104312944A - Microbial preparation, and preparation method and application thereof - Google Patents

Microbial preparation, and preparation method and application thereof Download PDF

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Publication number
CN104312944A
CN104312944A CN201410495939.XA CN201410495939A CN104312944A CN 104312944 A CN104312944 A CN 104312944A CN 201410495939 A CN201410495939 A CN 201410495939A CN 104312944 A CN104312944 A CN 104312944A
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preparation
oil
bacterium
microbial preparation
gs3c
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党志
李静华
郭楚玲
何丽媛
易筱筠
卢桂宁
杨琛
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South China University of Technology SCUT
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South China University of Technology SCUT
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
    • B09C—RECLAMATION OF CONTAMINATED SOIL
    • B09C1/00—Reclamation of contaminated soil
    • B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2103/00—Nature of the water, waste water, sewage or sludge to be treated
    • C02F2103/34—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32
    • C02F2103/36—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the manufacture of organic compounds
    • C02F2103/365—Nature of the water, waste water, sewage or sludge to be treated from industrial activities not provided for in groups C02F2103/12 - C02F2103/32 from the manufacture of organic compounds from petrochemical industry (e.g. refineries)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

The invention discloses a microbial preparation, and apreparation method and an application thereof. The microbial preparation comprises three strains of alkane degrading bacteria GS3C, phenanthrene degrading bacteria GY2B and pyrene degrading bacteria GP3B, or comprise two strains of the GS3C and the GP3B; and the GS3C, the GY2B and the GP3B are preserved in China Center for Type Culture Collection, and have the preservation numbers of CCTCC NO:M207169, CCTCC NO:M206019 and CCTCC NO:M207167 respectively. The microbial preparation provided by the invention can be used in the bioremediation of petroleum contaminated water and soil; and the microbial preparation can degrade petroleum in water to realize a petroleum removal rate of 69.2%, and has strong degradation ability on alkanes and aromatic compounds in petroleum. The mixed bacteria Gmix3 are immobilized with non-sterilized corn straws as a carrier, and are cultured in water for 3d to realize a petroleum removal rate of 98.2%.

Description

A kind of microbial preparation and its preparation method and application
Technical field
The invention belongs to environmental organic pollutant biologic treating technique field, be specifically related to a kind of hybrid bacterial strain microbial preparation and method for making thereof and application.
Background technology
Oil is human being's production and indispensable important energy source and industrial raw material in life.Along with the widespread use of oil, the leakage in the process such as exploitation, smelting, processing, transport, storage of oil, and the discharge of oily(waste)water refuse, cause serious environmental pollution.A large amount of oil and processed goods thereof enter water body, cause water hypoxia, destroy ecosystem balance, if enter soil, then spoiled soil structure, affect soil permeability, cause soil fertility to decline, directly can also damage plant root simultaneously, hinder breathing and the absorption of root, affect plant growth even dead, thus directly affect human being's production and life.Polycyclic aromatic hydrocarbons (PAHs) in oil has serious carcinogenesis, mutagenesis, aberration inducing " three cause " effect, can accumulate, not only affect grain quality and output in crop, also and human health biological by food chain harm.Therefore, administering petroleum-contaminated water and soil, is pendulum urgent problem in face of us.
The advantages such as bioremediation technology is low with processing cost, non-secondary pollution, become the important method of administering petroleum pollution gradually.The complex mixture that oil is made up of stable hydrocarbon, compound fragrant hydrocarbon and a small amount of bituminous matter, resene etc., and single strain often can only one or more limited petroleum components of metabolism.Symbiosis between mixed bacterial, association's equivalent effect can make mixed bacterium to the degradation effect of oil often higher than single strain.At present to the research of community construction just be separated the single bacterial classification obtained simple and relax be combined into flora, be difficult to reach the set goal.Therefore, the synergy between the dominant bacteria utilizing degradable oil different components (alkane and aromatic hydrocarbon), optimum combination can go out efficient degradation flora.
Add efficient degrading bacteria when repairing on the spot, usually be subject to the interference of various factors (between soil physico-chemical property, environmental factors, living species competition and predation etc.), cause adding that bacterium quantity reduces rapidly, non-activity or death, affect the repairing effect to oil.
Immobilized microorganism technique adopts physics or chemical means to be fixed on porous carrier by free cell (microorganism), make microorganism highly dense and keep its bioactive functions, reduce the interference of extraneous unfavorable factor to microorganism, effectively can improve the deficiency of traditional biological recovery technique, be conducive to the biological restoration on the spot to petroleum pollution.
Summary of the invention
The shortcoming that the object of the invention is to overcome prior art, with not enough, provides the construction process of a kind of high efficient petroleum degrading bacteria group and process for fixation thereof and application.The mix bacterium agent the present invention is directed to above-mentioned existing petroleum pollution bioremediation technology institute problems faced, design mechanism of degradation is complementary, act synergistically, and being fixed, carry out reparation to the water body of petroleum pollution and soil and apply.
The object of the invention is achieved through the following technical solutions:
A kind of microbial preparation, is formed by alkane degradation bacterial GS3C (Burkholderia cepacia), luxuriant and rich with fragrance degradation bacteria GY2B (Sphingomonas sp.) and pyrene degradation bacteria GP3B (Pandoraea pnomenusa) three bacterial strains or is made up of GS3C and GP3B two bacterial strain wherein; Described GS3C is preserved in China typical culture collection center on November 1st, 2007, and (be called for short CCTCC, address: wuchang, wuhan Luo Jiashan, Wuhan University, postcode: 430072), deposit number is CCTCC NO:M207169; GY2B is preserved in China typical culture collection center on February 24th, 2006, and deposit number is CCTCCNO:M206019; GP3B is preserved in China typical culture collection center on October 30th, 2006, and deposit number is CCTCC NO:M207167.
Described bacterial strain is mixed and made into immobilized microorganism preparation.
The preparation of described immobilized microorganism preparation: carry out enlarged culturing by after described bacterial strain mixing, the scale-up medium obtained carries out mixed culture with carrier again, being i.e. fixed microbial preparation, and described carrier is agricultural wastes.
The preparation method of mentioned microorganism preparation, comprises the following steps:
(1) single strain gradient domestication in oil substratum is cultivated;
(2) single strain of taming utilizes enrichment medium enlarged culturing, centrifuge washing, is the bacteria suspension of 0.5 with sterilized water configuration OD600;
(3) the bacteria suspension equal-volume of single strain is mixed, namely obtain microbial preparation.Above-mentioned preparation method also comprises the steps:
(a) by microbial preparation by volume percentage ratio 2% ~ 6% amount access 2000mg/L oil substratum, regulate pH be 7,30 DEG C cultivate 5 ~ 7 days, obtain the scale-up medium of mixed bacterium;
(b) by scale-up medium and carrier in mass ratio 1:1 ~ 3:5 mix, Dual culture 3 days at 30 DEG C, i.e. obtained immobilized microorganism preparation.
The compound method of described oil substratum: 5.0mL phosphate buffered saline buffer, concentration 22.5gL -1mgSO 4aqueous solution 3.0mL, concentration 36.4gL -1caCl 2aqueous solution 1.0mL, concentration 0.25gL -1feCl 3aqueous solution 1.0mL, 1.0mL trace element solution, 2000mg oil, adjust pH to be 7.0, distilled water is settled to 1L;
Described enrichment medium is: 10g peptone, 5g extractum carnis, 5g NaCl, and adjust pH to be 7.0, distilled water is settled to 1L.
The formula of described phosphate buffered saline buffer is 8.5gL -1kH 2pO 4, 21.75gL -1k 2hPO 4h 2o, 33.4gL -1na 2hPO 412H 2o, 5.0gL -1nH 4cl;
The formula of described trace element solution is 39.9mgL -1mnSO 4h 2o, 42.8mgL -1znSO 4h 2o, 34.7mgL -1(NH 4) 6mo 7o 244H 2o, described oil is lightweight oil, and density is: 0.863g (cm 3) -1.
Described carrier is through following pre-treatment: carrier is maize straw, rice straw or wood chip, and through air-dry, Mechanical Crushing, crosses the screen cloth in 0.2-0.9mm aperture, be washed to colourless, dries more than 12h for 60 DEG C.
Described gradient domestication: the concentration increasing oil is successively: 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L; Meanwhile, the corresponding original carbon source concentration of single strain (the wherein n-hexadecane of GS3C bacterium: 1500mg/L, 1000mg/L, 500mg/L and 0 is reduced gradually; The phenanthrene of GY2B bacterium: the pyrene of 100mg/L, 50mg/L, 10mg/L and 0, GP3B bacterium: 15mg/L, 10mg/L, 5mg/L and 0).
The application of described microbial preparation in petroleum-contaminated water and soil organisms are repaired.The addition of microbial preparation is 2 ~ 6% (w/w).In application in soil organisms is repaired, preferably, also add the amount of surfactant of 2 ~ 6% (w/w) and/or the inorganic composite fertilizer dosage of 0.5 ~ 1% (w/w) simultaneously.Described tensio-active agent is anion-nonionic surfactant SDS-TX100, and mol ratio is 2:1, and in the annex solution of configuration, TX100 concentration is 2.9mmol/L, SDS concentration is 5.8mmol/L.
Described alkane degradation bacterial GS3C, its original carbon source is n-Hexadecane; Luxuriant and rich with fragrance degradation bacteria GY2B, its original carbon source is luxuriant and rich with fragrance; Pyrene degradation bacteria GP3B, its original carbon source is pyrene.
The present invention is relative to the beneficial effect of prior art and advantage:
(1) microbial preparation provided by the present invention is tamed and the high efficient petroleum degrading bacteria group of recombination to construct through oil by alkane degradation bacterial GS3C, luxuriant and rich with fragrance degradation bacteria GY2B and pyrene degradation bacteria GP3B, the dominant strain optimum combination constructing function flora of degradable oil different components, can more effectively and widely degraded oil component; Its optimum mixture ratio is 1:1:1, and top condition is: temperature 30 DEG C, initial pH value 7.0, inoculum size 4%, oil starting point concentration 2000mg/L, degraded oil 5d in water body, its clearance reaches 69.2%, and has stronger degradation capability to the alkane in oil and arene compounds.With unsterilised maize straw for carrier fixes mixed bacterium G mix3, water body is cultivated the clearance of 3d to oil and is reached 98.2%.By immobilization mix bacterium agent G mix3adding petroleum pollution concentration for 3 times with 2% (w/w) point is oil can be removed 53.0% in the soil of 13754.5mg/kg 60 days.
(2) fixation support utilized is cheap; waste reclaimation; after adding soil; not only can protect and stabilised microorganism; and bulk soil makes more polyoxy molecule enter; absorbed portion organism fully contacts with microorganism, thus is conducive to each component of microbiological deterioration soil PetroChina Company Limited., realizes the biological restoration to oil-polluted soils.
Accompanying drawing explanation
The GC-MS spectrogram of each single strain degraded oil of Fig. 1;
The GC-MS spectrogram of the mixed bacterium degraded oil of Fig. 2 combination;
The high efficient petroleum degrading bacteria group G that Fig. 3 builds mix3the fragment ion figure of degraded oil;
The high efficient petroleum degrading bacteria group G that Fig. 4 builds mix3optimum growh and degradation condition;
The high efficient petroleum degrading bacteria group G that Fig. 5 builds mix3the kinetic curve figure of degraded water body PetroChina Company Limited.;
The high efficient petroleum degrading bacteria group G that Fig. 6 builds mix3gC-MS spectrogram in degraded oil process;
Fig. 7 immobilized by stalk G mix3with non-immobilization G mix3to the degradation efficiency comparison diagram of oil;
The high efficient petroleum degrading bacteria group G that Fig. 8 builds mix3and the electron micrograph of immobilized microbial inoculum, the non-sterilizing maize straw of a, b sterilizing maize straw, the mixed bacterium G before c immobilization mix3, d maize straw immobilization G mix3, e maize straw and G mix3simple mixing;
The high efficient petroleum degrading bacteria group G that Fig. 9 builds mix3immobilization preparation remove the kinetic curve figure of water body PetroChina Company Limited..
Embodiment
Below in conjunction with drawings and Examples, the present invention is described further, but be not construed as limiting the invention.
Embodiment 1
Optimize optimum strain combination
Strain combinations optimization comprises the following steps:
A) the gradient domestication in minimal medium of petroleum component degraded single strain is cultivated; Described gradient domestication: increase successively carbon source-oil-concentration be: 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L; Meanwhile, the corresponding original carbon source concentration of single strain is reduced gradually, the wherein n-hexadecane of GS3C bacterium: 1500mg/L, 1000mg/L, 500mg/L and 0; The phenanthrene of GY2B bacterium: the pyrene of 100mg/L, 50mg/L, 10mg/L and 0, GP3B bacterium: 15mg/L, 10mg/L, 5mg/L and 0;
The compound method of described minimal medium: 5.0mL phosphate buffered saline buffer (8.5gL -1kH 2pO 4, 21.75gL -1k 2hPO 4h 2o, 33.4gL -1na 2hPO 412H 2o, 5.0gL -1nH 4cl), 3.0mL MgSO 4the aqueous solution (22.5gL -1), 1.0mL CaCl 2the aqueous solution (36.4gL -1), 1.0mL FeCl 3the aqueous solution (0.25gL -1), 1.0mL trace element solution (39.9mgL -1mnSO 4h 2o, 42.8mgL -1znSO 4h 2o, 34.7mgL -1(NH 4) 6mo 7o 244H 2o), adjust pH to be 7.0, distilled water is settled to 1L.
Described oil is lightweight oil, and density is: 0.863g (cm 3) -1.
The single strain of b) taming utilizes enrichment medium enlarged culturing, centrifuge washing, is the bacteria suspension of 0.5 with sterilized water configuration OD600; Described enrichment medium: 10g peptone, 5g extractum carnis, 5g NaCl, 1L distilled water, adjusts pH to be 7.0;
C) bacteria suspension of single strain is pressed the various combination equal-volume mixing described in table 1, respectively by the oil substratum (adding the oil of 2000mg/L in above-mentioned minimal medium) of the total amount access 2000mg/L of 4% (percent by volume), pH is regulated to be 7,30 DEG C of cultivations, measure it after 5 days to clearance and the petroleum component removal situation of oil, to obtain the mix bacterium agent of degradation effect the best.
The bacterial strain 5 days of various combination is as shown in table 1 to the clearance of oil, and the bacterial strain of various combination removes situation as shown in FIG. 1 to 3 to petroleum component in 5 days.
Result is as follows:
(1) bacterial strain 5 days clearances to oil of various combination
Four kinds of single strains (wherein: GP3A is Pseudomonas, China typical culture collection center is preserved on October 30th, 2006, deposit number is CCTCC No:M207166) can be that sole carbon source carries out growth and breeding with oil after long-term domestication, all more than 30% is reached to the clearance of oil.
But, after arbitrarily combining the single strain of oil can be utilized, the degradation effect not necessarily had.The degradation effect of bacterial strain to oil of some combination is poorer than single strain, but the mixed bacterium group G3 built and G8 best results, improve nearly 30% than the clearance of single strain, respectively called after G mix2and G mix3.As shown in table 1.High efficient petroleum degrading bacteria group G mix2and G mix3contain the bacterial strain (alkane degradation bacterial GS3C) of the alkane derivative that can utilize in petroleum component and the bacterial strain (luxuriant and rich with fragrance degradation bacteria GY2B and pyrene degradation bacteria GP3B) of arene compounds, these bacterium, by Co metabolism effect, can produce synergistic effect to improve the removal to oil in degradation process.
Table 1
(2) the removal situation to petroleum component in the bacterial strain 5 days of various combination
GS3C can remove the straight-chain paraffin compound in oil substantially, and GY2B has good degradation effect to arene compounds.The degradation effect of GP3B to alkanes and arene compounds is general, as shown in Figure 1.By good for degradation effect mixed bacterium group G1, G3 (G mix2), G8 (G mix3) and G11 nutrient solution in Residual oil carry out GC-MS analysis, as shown in Figure 2, these four groups of mixed bacterium all include alkane degradation bacterial GS3C, the straight-chain paraffin compounds in oil can be removed substantially, remain the branched paraffin of some difficult degradations.Simultaneously again containing aromatic hydrocarbons degradation bacteria strains, be conducive to the completely removal of mixed bacterium to oil.Wherein G8 (G mix3) the highest to the total removal rate of oil, and remarkable to the degradation effect of arene compounds, 99.22% is reached to the clearance of naphthalene homologue, 86.02% is reached to the clearance of luxuriant and rich with fragrance homologue, as shown in Figure 3.Mixed bacterium G8 (G mix3) in include Sphingol single-cell GY2B arene compounds being had to strong degradation capability, in mixed system, GY2B has still played its degradation characteristic.In sum, alkanes degradation bacteria GS3C and aromatic hydrocarbons degradation bacteria GY2B and GP3B can symbiosis and alkanes in cooperative degradation of petroleum component and arene compounds, and what be conducive to petroleum component is degradable.
Embodiment 2
High efficient petroleum degrading bacteria group G mix3optimum growh and degradation condition
Test high efficient petroleum degrading bacteria group G respectively mix3in the different ratios of three strain bacterium, envrionment temperature, the initial pH of nutrient solution, inoculum size and difference initial petroleum concentration on the impact of the clearance of oil and the increment of bacterium, optimization high efficient petroleum degrading bacteria group G mix3best degradation condition be: the optimum mixture ratio of three strain bacterium is 1:1:1 (OD600 is the mother liquor of 0.5); Optimum growing condition is: temperature 30 DEG C, nutrient solution initial pH value 7.0, inoculum size 4%, oil starting point concentration 2000mg/L.At optimum conditions, degraded oil 5d in water body, its clearance reaches 69.2%, and has stronger degradation capability to the alkane in oil and arene compounds.As shown in Figure 4.
Embodiment 3
High efficient petroleum degrading bacteria group G mix3degraded oil over time.
Mixed bacterium G mix3in Initial stage of culture poor growth, after entering logarithmic phase, the degradation effect of oil is progressively strengthened.During 4d, the increment of mixed bacterium is increased sharply, and reaches 68.04% to the clearance of oil.5th celestial stone oil removing rate reaches maximum value 69.2%.After this microorganism growth enters stationary phase and decline phase, and aged bacterium is increased, and children bacterium in age reduces, and the microbial activity in reaction system weakens, and remaining petroleum component is more difficult is biodegradable, and the degradation rate of mixed bacterium to oil is more and more lower.As shown in Figure 5.
GC-MS collection of illustrative plates shows, as shown in Figure 6, along with the increase of degradation time, and mixed bacterium G mix3more and more obvious to the removal effect of oil.When degrading the 1st day initial stage, mixed bacterium G mix3not obvious to oil removal effect.During by the 3rd day, in spectrogram, advantage peak is pristane peak (pristane) (35.30min) and phytane peak (40.26min), calculate clearance by total peak area and reach 62.48%, the straight-chain paraffin compounds of major part carbon number C ﹤ 20 is removed, and the degradation effect of mixed bacterium to long chain alkane compound is poor.When the 5th day, straight-chain paraffin compounds is substantially removed, and clearance reaches 83.15%, remains the branched alkane hydro carbons of some difficult degradations as pristane, phytane etc.After this along with the increase of degraded number of days, the degradation rate of mixed bacterium to oil is more and more lower.
Embodiment 4
High efficient petroleum degrading bacteria group G mix3immobilization preparation and not immobilized contrast and electron microscope structures thereof.
The preparation of immobilized microorganism preparation, comprises the following steps:
(1) the gradient domestication in oil substratum of petroleum component degraded single strain is cultivated; Described gradient domestication: the concentration increasing oil is successively: 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L; Meanwhile, the corresponding original carbon source concentration of single strain is reduced gradually, the wherein n-hexadecane of GS3C bacterium: 1500mg/L, 1000mg/L, 500mg/L and 0; The phenanthrene of GY2B bacterium: the pyrene of 100mg/L, 50mg/L, 10mg/L and 0, GP3B bacterium: 15mg/L, 10mg/L, 5mg/L and 0;
The compound method of described oil substratum: 5.0mL phosphate buffered saline buffer (8.5gL -1kH 2pO 4, 21.75gL -1k 2hPO 4h 2o, 33.4gL -1na 2hPO 412H 2o, 5.0gL -1nH 4cl), 3.0mLMgSO 4the aqueous solution (22.5gL -1), 1.0mL CaCl 2the aqueous solution (36.4gL -1), 1.0mL FeCl 3the aqueous solution (0.25gL -1), 1.0mL trace element solution (39.9mgL -1mnSO 4h 2o, 42.8mgL -1znSO 4h 2o, 34.7mgL -1(NH 4) 6mo 7o 244H 2o), 2000mg oil, adjust pH to be 7.0, distilled water is settled to 1L.
Described oil is lightweight oil, and density is: 0.863g (cm 3) -1.
(2) single strain of taming utilizes enrichment medium enlarged culturing, centrifuge washing, is the bacteria suspension of 0.5 with sterilized water configuration OD600; Described enrichment medium: 10g peptone, 5g extractum carnis, 5gNaCl, 1L distilled water, adjusts pH to be 7.0;
(3) the bacteria suspension equal-volume of single strain is mixed, be high-efficiency microorganism preparation of the present invention;
(4) enlarged culturing: by the oil substratum of high-efficiency microorganism preparation by the amount access 2000mg/L of 4% (percent by volume), regulate pH to be 7,30 DEG C and cultivate 5 days, the scale-up medium of high efficient petroleum degrading bacteria group;
(5) immobilization preparation: carrier (maize straw/rice straw) is through air-dry, and Mechanical Crushing, crosses the screen cloth in 0.2-0.9mm aperture, tap water, to colourless, dries more than 12h for 60 DEG C; By above-mentioned scale-up medium and carrier (maize straw/rice straw) in mass ratio 3:5 mix, Dual culture 3 days at 30 DEG C, is the immobilization preparation of high efficient petroleum degrading bacteria group.
(6) to add in oil substratum (oil starting point concentration is for 2000mg/L) with the microbial inoculum amount of 20 ~ 30g/L (carrier dry weight) and with the maize straw of equal in quality, rice straw respectively with high efficient petroleum degrading bacteria group G mix3simple mixing is made into the mixture of carrier and bacterium liquid, to only have the G of unbound state mix3for contrast, be placed in 30 DEG C, rotating speed is the shaking table shaking culture 3d of 180r/min, survey its oil clearance.
As shown in Figure 7, straw/maize straw immobilization mixed bacterium G is adopted mix3effect higher than the free bacterium G of non-immobilization mix3, also higher than straw/maize straw and G mix3simple mixtures.Straw/maize straw has certain dissemination to oil, oil can also be adsorbed onto stalk surface simultaneously.Only add the oil substratum of stalk, oil is all distributed in solution and forms a large amount of oil/water interfaces, is conducive to the biological degradation of oil on interface.Through immobilized mixed bacterium G mix3show very strong oil removal effect, than not immobilized free mixed bacterium G mix3improve nearly 40%, rice straw immobilization G mix3the oil clearance of the 3rd day reaches 90% ~ 95%, maize straw immobilization G mix3the oil clearance of the 3rd day reaches 95% ~ 98%.Mixed bacterium is after immobilization, a large amount of microorganism is adsorbed in the space in stalk and forms high density flora, and in substratum, also having a small amount of microorganism suspended, two portions microorganism is degraded to oil simultaneously, and the removal effect of oil is greatly improved.Whether stalk also has a certain impact through the removal effect of sterilising treatment to oil.Unsterilised effect is all good than the effect of sterilizing.Straw-like materials generally containing have an appointment 16% ~ 25% xylogen, it is cellulosic surrounding matrix, play give fibre machinery intensity and protection Mierocrystalline cellulose exempt from destroyed effect.But be 120 DEG C in temperature, under pressure maintains the high-temperature high-pressure state of 0.1 ~ 0.15MPa, sterilizing is after 30 minutes, and partial lignin receives the destruction of high-temperature steam, and the adsorption potential on stalk reduces, the adsorptive power of oil and mixed bacterium is declined, have impact on the removal effect to oil.
Shown in scanning electron microscope diagram 8, unpasteurized stalk surface causes part cracked through crusher, expose the hollow cellulose pipe of stalk inner tight arrangement, xylogen in stalk is not destroyed, still can support the hollow cellulose pipe of multi-disc Rotating fields in stalk, make stalk have larger specific surface area, more adsorption site, be conducive to adsorbing mixed bacterium G mix3carry out surface adsorption immobilization, the hollow cellulose pipe in stalk also helps the diffusion of substrate and meta-bolites, provides enough spaces and oxygen for maintaining the metabolism of microorganism normal physiological.Stalk and G mix3mixture because of not being fixed, the microbial biomass of stalk surface adsorption is less and adsorb insecure, is easily rinsed down.Through immobilized mixed bacterium G mix3between the gap that can be adsorbed on the inner and stalk of hollow cellulose pipe, the height of mixed bacterium cell density obviously in non-immobilization, and also mixed bacterium becomes dense growth, and thalli morphology homogeneous surface is smooth.
In a word, immobilized by stalk mixed bacterium G is adopted mix3effect higher than the free bacterium G of non-immobilization mix3, also higher than stalk and G mix3simple mixtures.Unsterilised stalk fixes mixed bacterium G as carrier mix3after, both make use of stalk to the dispersion of oil and adsorption, taken full advantage of again the biological degradation of mixed bacterium to oil, promoted the removal effect of oil, can more effective oil have been removed from environment.
Embodiment 5
High efficient petroleum degrading bacteria group G mix3immobilization preparation is to the degraded situation of water body PetroChina Company Limited..
(oil starting point concentration is for 2000mg/L) in oil substratum is added with the immobilized bacterium dosage of the high efficient petroleum degrading bacteria group of 20 ~ 30g/L (carrier dry weight), be placed in 30 DEG C, rotating speed is the shaking table shaking culture of 180r/min, its oil clearance is surveyed in different time points sampling.
As shown in Figure 9, maize straw immobilization G mix3the clearance of oil is increased gradually along with the increase of time.After degraded 1d, 84.39% is reached to the clearance of oil, specific ionization mixed bacterium G mix3clearance when 1d improves nearly 60%, significantly enhances the degradation rate of mixed bacterium.After degraded 3d, 93.81% is reached to the clearance of oil, after this along with the removal effect of increase to oil of time tends towards stability.For maize straw, but be not limited only to maize straw.
Embodiment 6
High efficient petroleum degrading bacteria group G mix3immobilization preparation is to the degraded situation of soil PetroChina Company Limited..
Adopt orthogonal, utilize high efficient petroleum degrading bacteria group G mix3immobilization preparation carries out reparative experiment, by G to oil-polluted soils mix3immobilization preparation adds total amount, microbial inoculum adds number of times, dosage of surfactant and inorganic composite fertilizer amount are set to four factors, each factor three levels.Experiment soil is actual contaminated soil near certain refinery, and pH is 4.1 ~ 4.3, and it is 116:4:1 that organic content is about 4%, C:N:P, belongs to sandy clay loam.Experiment is carried out in the basin alms bowl being of a size of 170mm/120mm/150mm, and the soil of each basin alms bowl dress 1.5kg, is placed in actual environment condition: temperature 27 DEG C-34 DEG C; Relative humidity 40%-70%.High efficient petroleum degrading bacteria group G mix3immobilization preparation contains total bacterium amount about 2.98 × 10 8c.f.u./g carrier (be wood chip herein, but be not only confined to wood chip).Microbial inoculum add time point be respectively initially, the 7th day and the 14th day.Tensio-active agent is anion-nonionic surfactant SDS-TX100, and mol ratio is 2:1, and in the annex solution of configuration, TX100 concentration is 2.9mmol/L, SDS concentration is 5.8mmol/L.Inorganic composite fertilizer is En Taike stability durable composite fertilizer, nitrogen phosphoris and potassium fertilizer ratio: 22:7:11, total nutrient >=40% (containing nitric nitrogen), belong to potassium sulfate compound fertilizer, three point two of corresponding dosage grinds the rear soil that directly adds as slow-release fertilizer, and remaining 1/3rd water-soluble solution inject soil as quick-acting fertilizer.Every 2 ~ 3 days plowed soils about moisturizing to 20%.After 2 months, measure the change of total petroleum hydrocarbon.
As shown in table 2, oil removal effect optimal conditions are the oil degradation flora G of 2% (w/w) mix3immobilization preparation divides 3 times and adds, and adds the SDS-TritonX-100 (mol ratio is 2:1) of 6% (v/w), within 60 days, the total petroleum hydrocarbon concentration in acid soil can be reduced to 6464.6mg/kg from 13754.5mg/kg, eliminate 53%.When the C:N:P in soil is 116:4:1, without the need to adding inorganic composite fertilizer, because inorganic composite fertilizer contains certain salt component, be unfavorable for that microbial growth is bred.Preparation dosage and add number of times all has significance impact on the oil removed in soil.
Table 2
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.

Claims (10)

1. a microbial preparation, it is characterized in that, this microbial preparation is formed by alkane degradation bacterial GS3C (Burkholderia cepacia), luxuriant and rich with fragrance degradation bacteria GY2B (Sphingomonas sp.) and pyrene degradation bacteria GP3B (Pandoraea pnomenusa) three bacterial strains or is made up of GS3C and GP3B two bacterial strain wherein; Described GS3C, GY2B, GP3B are in China typical culture collection center preservation, and deposit number is respectively CCTCC NO:M207169, CCTCC NO:M206019, CCTCC NO:M207167.
2. microbial preparation according to claim 1, is characterized in that, described bacterial strain is mixed and made into immobilized microorganism preparation.
3. microbial preparation according to claim 2, it is characterized in that, the preparation of described immobilized microorganism preparation: carry out enlarged culturing by after described bacterial strain mixing, the scale-up medium obtained carries out mixed culture with carrier again, i.e. being fixed microbial preparation, described carrier is agricultural wastes.
4. the preparation method of microbial preparation described in any one of claims 1 to 3, is characterized in that, comprise the following steps:
(1) single strain gradient domestication in oil substratum is cultivated;
(2) single strain of taming utilizes enrichment medium enlarged culturing, centrifuge washing, is the bacteria suspension of 0.5 with sterilized water configuration OD600;
(3) the bacteria suspension equal-volume of single strain is mixed, namely obtain microbial preparation.
5. preparation method according to claim 4, is characterized in that, also comprises the steps:
(a) by microbial preparation by volume percentage ratio 2% ~ 6% amount access 2000mg/L oil substratum, regulate pH be 7,30 DEG C cultivate 5 ~ 7 days, obtain the scale-up medium of mixed bacterium;
(b) by scale-up medium and carrier in mass ratio 1:1 ~ 3:5 mix, Dual culture 3 days at 30 DEG C, i.e. obtained immobilized microorganism preparation.
6. preparation method according to claim 5, is characterized in that,
The compound method of described oil substratum: 5.0mL phosphate buffered saline buffer, concentration 22.5gL -1mgSO 4aqueous solution 3.0mL, concentration 36.4gL -1caCl 2aqueous solution 1.0mL, concentration 0.25gL -1feCl 3aqueous solution 1.0mL, 1.0mL trace element solution, 2000mg oil, adjust pH to be 7.0, distilled water is settled to 1L;
Described enrichment medium is: 10g peptone, 5g extractum carnis, 5g NaCl, and adjust pH to be 7.0, distilled water is settled to 1L.
7. preparation method according to claim 6, is characterized in that,
The formula of described phosphate buffered saline buffer is 8.5gL -1kH 2pO 4, 21.75gL -1k 2hPO 4h 2o, 33.4gL -1na 2hPO 412H 2o, 5.0gL -1nH 4cl;
The formula of described trace element solution is 39.9mgL -1mnSO 4h 2o, 42.8mgL -1znSO 4h 2o, 34.7mgL -1(NH 4) 6mo 7o 244H 2o, described oil is lightweight oil, and density is: 0.863g (cm 3) -1.
8. the preparation method according to any one of claim 4 ~ 7, is characterized in that, described carrier is through following pre-treatment: carrier is maize straw, rice straw or wood chip, through air-dry, Mechanical Crushing, crosses the screen cloth in 0.2-0.9mm aperture, be washed to colourless, dry more than 12h for 60 DEG C.
9. preparation method according to claim 8, is characterized in that, described gradient domestication: the concentration increasing oil is successively: 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L; Meanwhile, the corresponding original carbon source concentration of single strain is reduced gradually, the wherein n-hexadecane of GS3C bacterium: 1500mg/L, 1000mg/L, 500mg/L and 0; The phenanthrene of GY2B bacterium: the pyrene of 100mg/L, 50mg/L, 10mg/L and 0, GP3B bacterium: 15mg/L, 10mg/L, 5mg/L and 0.
10. the application of the microbial preparation described in any one of claims 1 to 3 in petroleum-contaminated water and soil organisms are repaired.
CN201410495939.XA 2014-09-24 2014-09-24 Microbial preparation, and preparation method and application thereof Pending CN104312944A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475144A (en) * 2017-04-11 2017-12-15 辽宁科技大学 A kind of Pandora bacterium and its application method
CN109052656A (en) * 2018-07-13 2018-12-21 中国地质科学院水文地质环境地质研究所 A kind of activity powdered carbon-degradation bacteria composition and groundwater remediation technology
CN110511890A (en) * 2019-08-23 2019-11-29 北京大学 A kind of medicine compatibility method of oil degradation microbial function complementation and its oil degradation flora for instructing compatibility
CN111440756A (en) * 2020-05-22 2020-07-24 西安文理学院 Method for treating petroleum sewage by using thermophilic bacteria

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107475144A (en) * 2017-04-11 2017-12-15 辽宁科技大学 A kind of Pandora bacterium and its application method
CN109052656A (en) * 2018-07-13 2018-12-21 中国地质科学院水文地质环境地质研究所 A kind of activity powdered carbon-degradation bacteria composition and groundwater remediation technology
CN109052656B (en) * 2018-07-13 2021-04-16 中国地质科学院水文地质环境地质研究所 Activated carbon powder-degrading bacterium composition and groundwater remediation technology
CN110511890A (en) * 2019-08-23 2019-11-29 北京大学 A kind of medicine compatibility method of oil degradation microbial function complementation and its oil degradation flora for instructing compatibility
CN111440756A (en) * 2020-05-22 2020-07-24 西安文理学院 Method for treating petroleum sewage by using thermophilic bacteria
CN111440756B (en) * 2020-05-22 2023-05-26 西安文理学院 Method for treating petroleum sewage by using thermophilic bacteria

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